Microorganisms and methods for the production of aniline

ABSTRACT

A non-naturally occurring microbial organism having an aniline pathway includes at least one exogenous nucleic acid encoding an aniline pathway enzyme expressed in a sufficient amount to produce aniline. The aniline pathway includes (1) an aminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a 4-aminobenzoate carboxylyase or (2) an anthranilate synthase and an anthranilate decarboxylase. A method for producing aniline, includes culturing these non-naturally occurring microbial organisms under conditions and for a sufficient period of time to produce aniline.

This application is a divisional application of U.S. patent applicationSer. No. 15/141,002, filed on Apr. 28, 2016, which is a continuation ofU.S. patent application Ser. No. 14/664,033, filed Mar. 20, 2015, nowabandoned, which is a continuation application of U.S. patentapplication Ser. No. 12/910,671, filed Oct. 22, 2010, now abandoned,which claims the benefit of priority of U.S. Provisional Application No.61/254,630, filed Oct. 23, 2009, the entire contents of eachapplication, which applications are incorporated herein by reference intheir entireties.

BACKGROUND OF THE INVENTION

This invention relates generally to in silico design of organisms andengineering of organisms, more particularly to organisms having anilinebiosynthesis capability.

Aniline is an organic compound with the formula C₆H₇N and is a precursorto numerous complex chemicals. Aniline is usually produced industriallyin two steps from benzene. First, benzene is nitrated using aconcentrated mixture of nitric acid and sulfuric acid at 50 to 60° C.,to provide nitrobenzene. In the second step, nitrobenzene ishydrogenated, typically at 600° C. in presence of a nickel catalyst togive aniline. In an alternative process, aniline is prepared from phenoland ammonia as described in U.S. Pat. No. 3,965,182. The phenol, inturn, is derived from the cumene process.

The main application of aniline is in the manufacture of polyurethane.Aniline also has value in the production of dyestuffs. In addition toits use as a precursor to dyestuffs, it is a starting-product for themanufacture of many drugs, such as paracetamol (acetaminophen, Tylenol).Currently, the largest market for aniline is preparation of methylenediphenyl diisocyanate (MDI), some 85% of aniline serving this market.Other uses include rubber processing chemicals (9%), herbicides (2%),and dyes and pigments (2%).

When polymerized, aniline can be used as a type of nanowire for use as asemiconducting electrode bridge in, for example, nano-scale devices suchas biosensors. These polyaniline nanowires can be doped in order toachieve certain semiconducting properties.

It is desirable to develop a method for production of aniline byalternative means that substitute renewable for petroleum-basedfeedstocks, while also using less energy- and capital-intensiveprocesses. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism having an aniline pathway that includes atleast one exogenous nucleic acid encoding an aniline pathway enzymeexpressed in a sufficient amount to produce aniline. The aniline pathwayincludes an aminodeoxychorismate synthase, an aminodeoxychorismatelyase, and a 4-aminobenzoate carboxylyase.

In some aspects, embodiments disclosed herein relate to a method forproducing aniline, that includes culturing a non-naturally occurringmicrobial organism having an aniline pathway that includes at least oneexogenous nucleic acid encoding an aniline pathway enzyme expressed in asufficient amount to produce aniline, under conditions and for asufficient period of time to produce aniline. The aniline pathwayincludes an aminodeoxychorismate synthase, an aminodeoxychorismatelyase, and a 4-aminobenzoate carboxylyase.

In some aspects, embodiments disclosed herein relate to a non-naturallyoccurring microbial organism having an aniline pathway that includes atleast one exogenous nucleic acid encoding an aniline pathway enzymeexpressed in a sufficient amount to produce aniline. The aniline pathwayincludes an anthranilate synthase and an anthranilate decarboxylase.

In some aspects, embodiments disclosed herein relate to a method forproducing aniline, that includes culturing a non-naturally occurringmicrobial organism having an aniline pathway that includes at least oneexogenous nucleic acid encoding an aniline pathway enzyme expressed in asufficient amount to produce aniline, under conditions and for asufficient period of time to produce aniline. The aniline pathwayincludes an anthranilate synthase and an anthranilate decarboxylase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the metabolic pathway to chorismate. E4P iserythrose-4-phosphate, PEP is phosphoenolpyruvate, DAHP is3-deoxy-D-arabino-heptulosonic acid-7-phosphate.

FIG. 2 shows metabolic pathways for the production of aniline. E4P iserythrose-4-phosphate, PEP is phosphoenolpyruvate, DAHP is3-deoxy-D-arabino-heptulosonic acid-7-phosphate.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed, in part, to the design and production ofcells and microbial organisms incorporating biosynthetic pathways forthe production of aniline. Enzymes useful for the production of anilinefrom the central metabolism precursors erythrose-4-phosphate (E4P) andphosphoenolpyruvate (PEP), via multiple routes, are depicted in FIG. 2.Such organisms can utilize renewable feedstocks, providing analternative to petroleum based aniline production. The maximumtheoretical yield of aniline from glucose as the carbon source is 0.857mole/mole glucose based on the equation 1 below.7C₆H₁₂O₆+6NH₃→6C₆H₅NH₂+6CO₂+30H₂O  equation 1

Engineering these pathways into a microorganism involves cloning anappropriate set of genes encoding a set of enzymes into a productionhost described herein, optimizing fermentation conditions, and assayingproduct formation following fermentation. To engineer a production hostfor the production of aniline, one or more exogenous DNA sequence(s) canbe expressed in a microorganism. In addition, the microorganism can haveendogenous gene(s) functionally disrupted, deleted or overexpressed.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having an aniline pathway that includes at least oneexogenous nucleic acid encoding an aniline pathway enzyme expressed in asufficient amount to produce aniline. The aniline pathway includes anaminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a4-aminobenzoate carboxylyase, as depicted in FIG. 2.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism having an aniline pathway that includes at least oneexogenous nucleic acid encoding an aniline pathway enzyme expressed in asufficient amount to produce aniline. The aniline pathway includes ananthranilate synthase and an anthranilate decarboxylase, as depicted inFIG. 2.

In some embodiments, the invention provides a method for producinganiline that includes culturing a non-naturally occurring microbialorganism having an aniline pathway. The pathway includes at least oneexogenous nucleic acid encoding an aniline pathway enzyme expressed in asufficient amount to produce aniline, under conditions and for asufficient period of time to produce aniline. In some embodiments, theaniline pathway includes an aminodeoxychorismate synthase, anaminodeoxychorismate lyase, and a 4-aminobenzoate carboxylyase. In otherembodiments the aniline pathway includes an anthranilate synthase and ananthranilate decarboxylase.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial organism's genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within an anilinebiosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism that the more than one exogenousnucleic acid refers to the referenced encoding nucleic acid orbiosynthetic activity, as discussed above. It is further understood, asdisclosed herein, that such more than one exogenous nucleic acids can beintroduced into the host microbial organism on separate nucleic acidmolecules, on polycistronic nucleic acid molecules, or a combinationthereof, and still be considered as more than one exogenous nucleicacid. For example, as disclosed herein a microbial organism can beengineered to express two or more exogenous nucleic acids encoding adesired pathway enzyme or protein. In the case where two exogenousnucleic acids encoding a desired activity are introduced into a hostmicrobial organism, it is understood that the two exogenous nucleicacids can be introduced as a single nucleic acid, for example, on asingle plasmid, on separate plasmids, can be integrated into the hostchromosome at a single site or multiple sites, and still be consideredas two exogenous nucleic acids. Similarly, it is understood that morethan two exogenous nucleic acids can be introduced into a host organismin any desired combination, for example, on a single plasmid, onseparate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two or moreexogenous nucleic acids, for example three exogenous nucleic acids.Thus, the number of referenced exogenous nucleic acids or biosyntheticactivities refers to the number of encoding nucleic acids or the numberof biosynthetic activities, not the number of separate nucleic acidsintroduced into the host organism.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having aniline biosyntheticcapability, those skilled in the art will understand with applying theteaching and guidance provided herein to a particular species that theidentification of metabolic modifications can include identification andinclusion or inactivation of orthologs. To the extent that paralogsand/or nonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

In some embodiments, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having an anilinepathway comprising at least one exogenous nucleic acid encoding ananiline pathway enzyme expressed in a sufficient amount to produceaniline, said aniline pathway comprising an aminodeoxychorismatesynthase, an aminodeoxychorismate lyase, and a 4-aminobenzoatecarboxylyase. In some embodiments, such a non-naturally occurringmicrobial organism can further include a DAHP synthase and in stillfurther embodiments, the non-naturally occurring microbial organism canfurther include a 3-dehydroquinate synthase.

In some embodiments, the non-naturally occurring microbial organismincludes two exogenous nucleic acids each encoding an aniline pathwayenzyme, while in other embodiments the non-naturally occurring microbialorganism includes three exogenous nucleic acids each encoding an anilinepathway enzyme. For example, in some embodiments, the non-naturallyoccurring microbial organism can include three exogenous nucleic acidsencoding an aminodeoxychorismate synthase, an aminodeoxychorismatelyase, and a 4-aminobenzoate carboxylyase.

In some embodiments, the non-naturally occurring microbial organism caninclude four exogenous nucleic acids each encoding an aniline pathwayenzyme. For example, a non-naturally occurring microbial organism havingfour exogenous nucleic acids can encode a DAHP synthase, anaminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a4-aminobenzoate carboxylyase. The DAHP synthase, which can be endogenousto the non-naturally occurring microbial organism, can be overexpressed,for example, by insertion of additional copies of the gene and/orthrough the use of exogenous regulatory genes and removing feedbackregulation by the aromatic amino acids.

In still further embodiments, the non-naturally occurring microbialorganism can include five exogenous nucleic acids each encoding ananiline pathway enzyme. For example, the non-naturally occurringmicrobial organism having five exogenous nucleic acids can encode a3-dehydroquinate synthase, a DAHP synthase, an aminodeoxychorismatesynthase, an aminodeoxychorismate lyase, and a 4-aminobenzoatecarboxylase. The 3-dehydroquinate synthase, which can be endogenous tothe non-naturally occurring microbial organism, can also beoverexpressed, for example, by insertion of additional copies of thegene and/or through the use of exogenous regulatory genes.

Moreover, any one or more of the other enzymes that are in a pathway enroute to chorismate, which can be endogenous in some embodiments, can beoverexpressed to increase the production of chorismate. These include,for example, a 3-dehydroquinate dehydratase (EC 4.2.1.10), a shikimatedehydrogenase (1.1.1.25), a quinate/shikimate dehydrogenase (1.1.1.282),a shikimate kinase (2.7.1.71), a3-phosphoshikimate-1-carboxyvinyltransferase (2.5.1.19), and achorismate synthase (4.2.3.5). These enzymes constitute the pathway formaking chorismate from DAHP in prokaryotes and most eukaryotes. Analternative pathway for formation of 3-dehydroquinate (the steps from3-dehydroquinate to chorismate are the same in all organisms, includingarachea) includes the following enzymatic steps: triosephosphateisomerase, frustose-1,6-bisphosphate aldolase,2-amino-3,7-dideoxy-D-threo-hept-6-ulosonate synthase, anddehydroquinate synthase.

In some embodiments, the non-naturally occurring microbial organismsdescribed above can have at least one exogenous nucleic acid which is aheterologous nucleic acid. Moreover, the non-naturally occurringmicrobial organisms described above, can be provided in a substantiallyanaerobic culture medium.

In some embodiments, the present invention also provides a non-naturallyoccurring microbial organism having an aniline pathway that includes atleast one exogenous nucleic acid encoding an aniline pathway enzymeexpressed in a sufficient amount to produce aniline, in which theaniline pathway includes an anthranilate synthase and an anthranilatedecarboxylase. Such a non-naturally occurring microbial organism canfurther include a DAHP synthase, as described above. In someembodiments, such a non-naturally occurring microbial organism canfurther include a 3-dehydroquinate synthase.

In some embodiments, this non-naturally occurring microbial includes twoexogenous nucleic acids each encoding an aniline pathway enzyme. Forexample, the two exogenous nucleic acids can encode an anthranilatesynthase and an anthranilate decarboxylase. In some embodiments themicrobial organism can include three exogenous nucleic acids eachencoding an aniline pathway enzyme. For example, the three exogenousnucleic acids can encode a DAHP synthase, an anthranilate synthase andan anthranilate decarboxylase. In some embodiments, the microbialorganism includes four exogenous nucleic acids each encoding an anilinepathway enzyme. For example, the four exogenous nucleic acids can encodea 3-dehydroquinate synthase, a DAHP synthase, an anthranilate synthaseand an anthranilate decarboxylase.

As described above, any one or more of the other enzymes that are in apathway en route to chorismate, which can be endogenous in someembodiments, can be overexpressed to increase the production ofchorismate. These include, for example, a 3-dehydroquinate dehydratase,a shikimate dehydrogenase, a quinate/shikimate dehydrogenase, ashikimate kinase, a 3-phosphoshikimate-1-carboxyvinyltransferase, and achorismate synthase.

In some embodiments, such non-naturally occurring microbial organismsdescribed above can include at least one exogenous nucleic acid is aheterologous nucleic acid. In some embodiments, such non-naturallyoccurring microbial organisms are in a substantially anaerobic culturemedium.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having an aniline pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of chorismateto 4-amino-4-deoxychorismate, 4-amino-4-deoxychorismate top-aminobenzoate, and p-aminobenzoate to aniline.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having an aniline pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product selected from the group consisting of chorismateto anthranilate, and anthranilate to aniline.

One skilled in the art will understand that these are merely exemplaryand that any of the substrate-product pairs disclosed herein suitable toproduce a desired product and for which an appropriate activity isavailable for the conversion of the substrate to the product can bereadily determined by one skilled in the art based on the teachingsherein. Thus, the invention provides a non-naturally occurring microbialorganism containing at least one exogenous nucleic acid encoding anenzyme or protein, where the enzyme or protein converts the substratesand products of an aniline pathway, such as that shown in FIG. 1.

While generally described herein as a microbial organism that containsan aniline pathway, it is understood that the invention additionallyprovides a non-naturally occurring microbial organism comprising atleast one exogenous nucleic acid encoding an aniline pathway enzymeexpressed in a sufficient amount to produce an intermediate of ananiline pathway. For example, as disclosed herein, an aniline pathway isexemplified in FIG. 1. Therefore, in addition to a microbial organismcontaining an aniline pathway that produces aniline, the inventionprovides a non-naturally occurring microbial organism comprising atleast one exogenous nucleic acid encoding an aniline pathway enzyme,where the microbial organism produces an aniline pathway intermediate,for example, DAHP, chorismate, anthranilate, 4-amino-4-deoxychorismate,or p-aminobenzoate.

It is understood that any of the pathways disclosed herein, as describedin the Examples and exemplified in the Figures, including the pathwaysof FIG. 1, can be utilized to generate a non-naturally occurringmicrobial organism that produces any pathway intermediate or product, asdesired. As disclosed herein, such a microbial organism that produces anintermediate can be used in combination with another microbial organismexpressing downstream pathway enzymes to produce a desired product.However, it is understood that a non-naturally occurring microbialorganism that produces an aniline pathway intermediate can be utilizedto produce the intermediate as a desired product.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

As shown in FIG. 1, the first step of an aniline pathway is analdol-type condensation that combines one molecule of E4P and onemolecule of PEP to form the intermediate, 3-deoxy-D-arabino-heptulosonicacid 7-phosphonate (DAHP). This enzyme is referred to as DAHP synthase,or equivalently, 2-dehydro-3-deoxyphosphoheptonate aldolase. Thisreaction (EC #2.5.1.54) is the first committed step in the shikimatepathway and is required for the biosynthesis of aromatic amino acids,folates, quinones and other secondary metabolites in bacteria, fungi andplants. DAHP synthases have been categorized into AroAI and AroAIIclasses (Wu et al., J. Biol. Chem. 281:4042-4048 (2006)). The formerclass comprises of mainly microbial proteins while the latter iscomprised of primarily plant proteins.

In Escherichia coli, the function is catalyzed by three genes: aroFGH.Each of these encodes for an isozyme and is feedback regulated by adifferent aromatic amino acid. In contrast, some other organisms, suchas Bacillus subtilis and Porphyromonas gingivalis are bifunctionalenzymes. The aroA gene encodes for DAHP synthase activity and aroQ geneencodes for chorismate mutase activity in B. subtilis. However, theseactivities can be separated by domain truncation ((Wu et al., J. Biol.Chem. 281:4042-4048 (2006)). The B. subtilis enzyme is sensitive to thedownstream intermediates, chorismate and prephanate. The DAHP synthasefrom Corynebacterium glutamicum is feedback sensitive to both,phenylalanine and tyrosine (Wu et al., J. Biol. Chem. 278:27525-27531(2003)).

These enzymes are metalloenzymes and their mechanisms of regulation arewell-understood by those skilled in the art. The crystal structures ofthe E. coli and S. cerevisiae DAHP synthases have been solved and revealstructures consisting of (β/α)₈ barrel. There are several enzymes thathowever, don't have regulatory domains and belong to organisms such asPyrococcus furiosus and Nostoc sp. Exemplary genes are summarized belowin Table 1.

TABLE 1 Gene GI number GenBank ID Organism aroF 16130522 NP_417092.1Escherichia coli K12 MG1655 aroG 16128722 NP_415275.1 Escherichia coliK12 MG1655 aroH 16129660 NP_416219.1 Escherichia coli K12 MG1655 aroA16080027 NP_390853.1 Bacillus subtilis aroA 34396967 AAQ66031.1Porphyromonas gingivalis W83 Aro4 6319726 NP_009808.1 Saccharomycescerevisiae aroG 1168513 P44303.1 Haemophilus influenza aroF 16765985NP_461600.1 Salmonella typhimurium aroG 21903376 P35170.2Corynebacterium glutamicum PF1690 18893851 AAL81814.1 Pyrococcusfuriosus alr3050 17132144 BAB74749 Nostoc sp

Chorismate is a known intermediate for aromatic amino acid biosynthesisin Gram positive, Gram-negative bacteria and in archaea. It is also aprecursor for the production of folic acid, ubiquinone, menaquinone andenterocholein in some microorganisms. DAHP can be converted into3-dehydroquinate and that can be subsequently converted into chorismatevia multiple well-known steps. In E. coli, DAHP can be converted into3-dehydroquinate by 3-dehydroquinate synthase. The synthase in E. coliis understood to catalyze an oxidation, a β-elimination, anintramolecular aldol condensation and a reduction (Frost et al.,Biochemistry 23:4470-4475 (1984); Maitra et al., J. Biol. Chem.253:5426-5430 (1978)). The enzyme requires catalytic amounts of NAD⁺ andCo²⁺ (Maitra et al., J. Biol. Chem. 253:5426-5430 (1978)). Enzymesuseful for the production of chorismate include, for example, a3-dehydroquinate dehydratase, a shikimate dehydrogenase, aquinate/shikimate dehydrogenase, a shikimate kinase, a3-phosphoshikimate-1-carboxyvinyltransferase, and a chorismate synthase,as described above

The conversion of chorismate into 4-amino-4-deoxychorismate can beaccomplished by aminodeoxychorismate synthase (EC#2.6.1.85), alsoreferred to as chorismate L-glutamine aminotransferase. In E. coli, thefunction is catalyzed by two genes, pabA and pabB. The pabA polypeptideis a conditional glutaminase which requires a 1:1 complex with pabB foractivity. The pabB enzyme uses the nascent ammonia released by thisreaction to transform chorismate to 4-amino-4-deoxychorismate (in thepresence of Mg²⁺). The pabB reaction is fully reversible. In the absenceof pabA, pabB utilizes NH₃ at significantly reduced rates (Roux andWalsh, Biochemistry 32:3763-3768 (1993); Roux and Walsh, Biochemistry31:6904-6910 (1992)).

A similar enzyme complex formed by pabA and pabB, catalyzes theconversion of chorismate into 4-amino-4-dexoychorismate in Streptomycesvenezuelae (Brown et al., Microbiology 142(pt 6): 1345-1355 (1996)).This organism is known to have more than one set of pabAB genes (Changet al., Microbiology 147:2113-2126 (2001)). The gene with theaforementioned function has been identified in Arabidopsis thaliana andSolanum lycopersicum also. The protein sequences of the PabA and PabBgenes of E. coli were used to isolate the cDNA encoding theaminodeoxychorismate synthase (ADCS) in Arabidopsis thaliana (Basset etal., Proc. Natl. Acad. Sci. U.S.A. 101:1496-1501 (2004)). The enzyme wasrecombinantly expressed in E. coli demonstrating the formation of4-amino-4-deoxychorismate. No feedback inhibition of the enzyme has beenreported for either p-aminobenzoate or folate. The corresponding genes,along with their GenBank ids are listed below in Table 2:

TABLE 2 pabAB 710438 AAB30312.1 Streptomyces venezuelae pabA 16131239NP_417819.1 Escherichia coli K12 MG1655 pabB 16129766 NP_416326.1Escherichia coli K12 MG1655 pabA 152972254 YP_001337400.1 Klebsiellapneumoniae pabB 152970875 YP_001335984.1 Klebsiella pneumoniae pabA118467576 YP_884448.1 Mycobacterium smegmatis pabB 118473035 YP_889684.1Mycobacterium smegmatis

In several organisms, the gene encoding for aminodeoxychorismate lyase(EC#4.1.3.38) is typically coupled with pabB and pabA to catalyze theconversion of aminodexoychorismate into p-aminobenzoate, with therelease of a pyruvate molecule. In both E. coli (Green et al., J.Bacteriol. 174:5317-5323 (1992); Green and Nichols, J. Biol. Chem.266:12971-12975 (1991)) and S. venezuelae, pabC catalyzes this reaction.Recently, 4-amino-4-deoxychorismate lyase was functionally characterizedin two more species of Streptomcyes, namely FR-008 and griseus (Zhang etal., Microbiology 155:2450-2459 (2009)). Aminodeoxychorismate synthaseand aminodeoxychorismate lyase are typically part of folate biosynthesisin most organisms and facilitate the conversion of chorismate intopara-aminobenzoate. Aminodeoxychorismate lyase is a pyridoxal-phosphatedependent protein. A putative enzyme has been found in A. thaliana((Basset et al., Proc. Natl. Acad. Sci. U.S.A. 101:1496-1501 (2004)) andB. subtilis (Schadt et al., J. Am. Chem. Soc. 131:3481-3493 (2009);Slock et al., J. Bacteriol. 172:7211-7226 (1990)), as part of the folateoperon. Some exemplary genes are shown below in Table 3:

TABLE 3 pabC 16129059 NP_415614.1 Escherichia coli K12 MG1655 pabC16077144 NP_387957.1 Bacillus subtilis pabC 29828105 NP_822739.1Streptomyces avermitilis pabC-1 224831591 AAQ82550.2 Streptomyces sp.FR-008 pabC-2 219879202 ACL50980.1 Streptomyces sp. FR-008

Anthranilate synthase (EC: 4.1.3.27), also known by the systematic namechorismate pyruvate-lyase (amino-accepting; anthranilate-forming) or bythe synonym glutamine amidotransferase, is the first step in thetryptophan synthesis pathway from chorismate. The formation ofanthranilate is accompanied by the transfer of an amine group fromglutamine and leading to the formation of glutamate. Pyruvate is alsoreleased during the reaction. In E. coli, this reaction is catalyzed bya tetrameric enzyme complex comprised of two monomers of TrpD and twomonomers of TrpE. TrpE on its own can carry out an alternate version ofthis reaction, using ammonium sulfate rather than glutamine as an aminodonor (Ito et al., Acta. Pathol. Jpn. 19:55-67 (1969); Ito and Yanofsky,J. Bacteriol. 97:734-742 (1969)). However, TrpD increases the affinityof TrpE for glutamine over TrpE alone. The enzyme is feedback regulatedby tryptophan. This feedback regulation is also observed for the enzymecomplex in the hyperthermophilic Sulfolobus solfataricus. The enzymecomplex from this organism has been expressed in E. coli (Tutino et al.,Biochem. Biophys. Res. Commun. 230:306-310 (1997)). The thermodynamicsof the reaction catalyzed by anthranilate synthase has been described inSalmonella typhimurium (Byrnes et al., Biophys. Chem. 84:45-64 (2000)).The subunits of the enzyme complex have also been described inThermotoga maritima (Kim et al., J. Mol. Biol. 231:960-981 (1993)). Asummary of these genes is shown below in Table 4.

TABLE 4 TrpD 16129224 NP_415779.1 Escherichia coli K12 MG1655 TrpE16129225 NP_415780.1 Escherichia coli K12 MG1655 TrpE 15897780NP_342385.1 Sulfolobus solfataricus TrpGD 15897781 NP_342386.1Sulfolobus solfataricus trpD 16765068 NP_460683.1 Salmonella typhimuriumtrpE 16765067 NP_460682.1 Salmonella typhimurium trpE 15642916NP_227957.1 Thermotoga maritima trpGD 15642915 NP_227956.1 Thermotogamaritima

The decarboxylation of p-aminobenzoate and anthranilate can be catalyzedby an aminobenzoate carboxylyase (McCullough et al., J. Am. Chem. Soc.79:628-630 (1957)). It has been indicated that the cell free enzymeobtained from E. coli 0111:B4 was capable of decarboxylating both ofthese molecules. The activity of the enzyme was found to be dependent onpyridoxal phosphate and iron (III). The conversion of p-aminobenzoate toaniline in some extracts of Mycobacteria has been described (Sloane etal., J. Biol. Chem. 193:453-458 (1951)). New strains have beenidentified that are capable of degrading aniline anaerobically (Kahng etal., FEMS Microbiol. Lett. 190:215-221 (2000); Schnell et al., Arch.Microbiol. 152:556-563 (1989)). These strains first carboxylate anilineto 4-aminobenzoate. In the strain, Desulfobacterium anilini, the rate ofaniline degradation is dependent on the presence of CO₂ in the medium.GC analysis of aniline culture supernatant of strain HY99 underanaerobic, denitrifying conditions showed the presence of4-aminobenzoate (Kahng et al., FEMS Microbiol. Lett. 190:215-221(2000)).

Numerous other studies have been conducted on decarboxylation ofaromatic compounds, primarily hydroxyl aromatics. For example, a4-hydroxybenzoate decarboxylase has been identified from the facultativeanaerobe, Enterobacter cloacae (Matsui et al., Arch. Microbiol.186:21-29 (2006)). The corresponding gene has been sequenced. The enzymehas been tested for activity on multiple substrates and was shown to beinduced by both 4-hydroxybenzoic acid and 4-aminobenzoic acid. Anotherdecarboxylase has been reported in Clostridium theromaceticum that canremove CO₂ from p-hydroxy benzoate (Hsu et al., J. Bacteriol.172:5901-5907 (1990)). The enzyme has broad substrate specificity andcan act on p-hydroxy benzoate with varied functional group substituentsat the meta-position. These include hydroxyl, chloro, fluoro, andmethoxy groups. The enzyme was not repressed by glucose or otherexternal energy sources. Klebsiella aerogens was also reported to beable to carry out non-oxidative decarboxylation of para-hydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate and3,4,5-trihydroxybenzoate (Grant et al., Antonie Van Leeuwenhoek35:325-343 (1969)). A reversible 4-hydroxybenzoate decarboxylase waspurified from Clostridium hydroxybenzoicum (now called Sedimentibacterhydroxybenzoicus). This enzyme is encoded by three clustered genes,shdB, C and D. The enzyme can act on both 4-hydroxybenzoate and3,4-dihydroxybenzoate. The enzyme activity was not affected by metalions or other cofactor (He et al., Eur. J. Biochem. 229:77-82 (1995)).Bacillus subtilis was recently demonstrated to have a hydroxyarylic aciddecarboxylase activity. Three genes bcdB, C, and D were cloned in E.coli and showed activity on 4-hydroxybenzoate and vanillate (Lupa etal., Can. J. Microbiol. 54:75-81 (2008)). These decarboxylases have beenreported in several other organisms (Lupa et al., Genomics 86:342-351(2005)) and gene candidates for some of these are listed below in Table5.

TABLE 5 shdB 67462197 AAY67850.1 Sedimentibacter hydroxybenzoicus shdC5739200 AAD50377.1 Sedimentibacter hydroxybenzoicus shdD 67462198AAY67851.1 Sedimentibacter hydroxybenzoicus 110331749 BAE97712.1Enterobacter cloacae bsdB 13124411 P94404.1 Bacillus subtilis bsdC6686207 P94405.1 Bacillus subtilis bsdD 239977069 C0H3U9.1 Bacillussubtilis STM292 16766227 NP_461842.1 Salmonella typhimurium LT2 STM292216766228 NP_461843.1 Salmonella typhimurium LT2 STM2923 16766229NP_461844.1 Salmonella typhimurium LT2 kpdB 206580833 YP_002236894.1Klebsiella pneumoniae 342 kpdC 206576360 YP_002236895.1 Klebsiellapneumoniae 342 kpdD 206579343 YP_002236896.1 Klebsiella pneumoniae 342pad1 15832847 NP_311620.1 Escherichia coli O157 yclC 15832846NP_311619.1 Escherichia coli O157 yclD 15832845 NP_311618.1 Escherichiacoli O157

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more anilinebiosynthetic pathways. Depending on the host microbial organism chosenfor biosynthesis, nucleic acids for some or all of a particular anilinebiosynthetic pathway can be expressed. For example, if a chosen host isdeficient in one or more enzymes or proteins for a desired biosyntheticpathway, then expressible nucleic acids for the deficient enzyme(s) orprotein(s) are introduced into the host for subsequent exogenousexpression. Alternatively, if the chosen host exhibits endogenousexpression of some pathway genes, but is deficient in others, then anencoding nucleic acid is needed for the deficient enzyme(s) orprotein(s) to achieve aniline biosynthesis. Thus, a non-naturallyoccurring microbial organism of the invention can be produced byintroducing exogenous enzyme or protein activities to obtain a desiredbiosynthetic pathway or a desired biosynthetic pathway can be obtainedby introducing one or more exogenous enzyme or protein activities that,together with one or more endogenous enzymes or proteins, produces adesired product such as aniline.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizopus oryzae, and the like. E. coli is aparticularly useful host organism since it is a well characterizedmicrobial organism suitable for genetic engineering. Other particularlyuseful host organisms include yeast such as Saccharomyces cerevisiae. Itis understood that any suitable microbial host organism can be used tointroduce metabolic and/or genetic modifications to produce a desiredproduct.

Depending on the aniline biosynthetic pathway constituents of a selectedhost microbial organism, the non-naturally occurring microbial organismsof the invention will include at least one exogenously expressed anilinepathway-encoding nucleic acid and up to all encoding nucleic acids forone or more aniline biosynthetic pathways. For example, anilinebiosynthesis can be established in a host deficient in a pathway enzymeor protein through exogenous expression of the corresponding encodingnucleic acid. In a host deficient in all enzymes or proteins of ananiline pathway, exogenous expression of all enzyme or proteins in thepathway can be included, although it is understood that all enzymes orproteins of a pathway can be expressed even if the host contains atleast one of the pathway enzymes or proteins. For example, exogenousexpression of all enzymes or proteins in a pathway for production ofaniline can be included, such as a 3-dehydroquinate synthase, a DAHPsynthase, an aminodeoxychorismate synthase, an aminodeoxychorismatelyase, and a 4-aminobenzoate carboxylyase or a 3-dehydroquinatesynthase, a DAHP synthase, an anthranilate synthase and an anthranilatedecarboxylase.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the anilinepathway deficiencies of the selected host microbial organism. Therefore,a non-naturally occurring microbial organism of the invention can haveone, two, three, four, five, up to all nucleic acids encoding theenzymes or proteins constituting an aniline biosynthetic pathwaydisclosed herein. In some embodiments, the non-naturally occurringmicrobial organisms also can include other genetic modifications thatfacilitate or optimize aniline biosynthesis or that confer other usefulfunctions onto the host microbial organism. One such other functionalitycan include, for example, augmentation of the synthesis of one or moreof the aniline pathway precursors such as chorismate, anthranilate,4-amino-4-deoxychorismate, and p-aminobenzoate.

Generally, a host microbial organism is selected such that it producesthe precursor of an aniline pathway, either as a naturally producedmolecule or as an engineered product that either provides de novoproduction of a desired precursor or increased production of a precursornaturally produced by the host microbial organism. For example,chorismate is produced naturally in a host organism such as E. coli. Ahost organism can be engineered to increase production of a precursor,as disclosed herein. In addition, a microbial organism that has beenengineered to produce a desired precursor can be used as a host organismand further engineered to express enzymes or proteins of an anilinepathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize aniline. In this specific embodiment it can beuseful to increase the synthesis or accumulation of an aniline pathwayproduct to, for example, drive aniline pathway reactions toward anilineproduction. Increased synthesis or accumulation can be accomplished by,for example, overexpression of nucleic acids encoding one or more of theabove-described aniline pathway enzymes or proteins. Over expression ofthe enzyme or enzymes and/or protein or proteins of the aniline pathwaycan occur, for example, through exogenous expression of the endogenousgene or genes, or through exogenous expression of the heterologous geneor genes. Therefore, naturally occurring organisms can be readilygenerated to be non-naturally occurring microbial organisms of theinvention, for example, producing aniline, through overexpression ofone, two, three, four, five, up to all nucleic acids encoding anilinebiosynthetic pathway enzymes or proteins. In addition, a non-naturallyoccurring organism can be generated by mutagenesis of an endogenous genethat results in an increase in activity of an enzyme in the anilinebiosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, an aniline biosynthetic pathway onto the microbial organism.Alternatively, encoding nucleic acids can be introduced to produce anintermediate microbial organism having the biosynthetic capability tocatalyze some of the required reactions to confer aniline biosyntheticcapability. For example, a non-naturally occurring microbial organismhaving an aniline biosynthetic pathway can comprise at least twoexogenous nucleic acids encoding desired enzymes or proteins, such asthe combination of aminodoxychorismate synthase and aminodeoxychorismatelyase, or aminodeoxychorismate lyase and 4-aminobenzoate carboxylase, oraminodeoxychorismate synthase and 4-amionbenzoate carboxylase, and thelike. Thus, it is understood that any combination of two or more enzymesor proteins of a biosynthetic pathway can be included in a non-naturallyoccurring microbial organism of the invention. Similarly, it isunderstood that any combination of three or more enzymes or proteins ofa biosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention, and so forth, as desired, so longas the combination of enzymes and/or proteins of the desiredbiosynthetic pathway results in production of the corresponding desiredproduct. Similarly, any combination of four, or more enzymes or proteinsof a biosynthetic pathway as disclosed herein can be included in anon-naturally occurring microbial organism of the invention, as desired,so long as the combination of enzymes and/or proteins of the desiredbiosynthetic pathway results in production of the corresponding desiredproduct.

In addition to the biosynthesis of aniline as described herein, thenon-naturally occurring microbial organisms and methods of the inventionalso can be utilized in various combinations with each other and withother microbial organisms and methods well known in the art to achieveproduct biosynthesis by other routes. For example, one alternative toproduce aniline other than use of the aniline producers is throughaddition of another microbial organism capable of converting an anilinepathway intermediate to aniline. One such procedure includes, forexample, the fermentation of a microbial organism that produces ananiline pathway intermediate. The aniline pathway intermediate can thenbe used as a substrate for a second microbial organism that converts theaniline pathway intermediate to aniline. The aniline pathwayintermediate can be added directly to another culture of the secondorganism or the original culture of the aniline pathway intermediateproducers can be depleted of these microbial organisms by, for example,cell separation, and then subsequent addition of the second organism tothe fermentation broth can be utilized to produce the final productwithout intermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, aniline. In theseembodiments, biosynthetic pathways for a desired product of theinvention can be segregated into different microbial organisms, and thedifferent microbial organisms can be co-cultured to produce the finalproduct. In such a biosynthetic scheme, the product of one microbialorganism is the substrate for a second microbial organism until thefinal product is synthesized. For example, the biosynthesis of anilinecan be accomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, aniline alsocan be biosynthetically produced from microbial organisms throughco-culture or co-fermentation using two organisms in the same vessel,where the first microbial organism produces an aniline intermediate andthe second microbial organism converts the intermediate to aniline.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce aniline.

Sources of encoding nucleic acids for an aniline pathway enzyme orprotein can include, for example, any species where the encoded geneproduct is capable of catalyzing the referenced reaction. Such speciesinclude both prokaryotic and eukaryotic organisms including, but notlimited to, bacteria, including archaea and eubacteria, and eukaryotes,including yeast, plant, insect, animal, and mammal, including human.Exemplary species for such sources include, for example, Escherichiacoli, Sedimentibacter hydroxybenzoicus, and Bacillus subtilis, as wellas other exemplary species disclosed herein or available as sourceorganisms for corresponding genes. However, with the complete genomesequence available for now more than 550 species (with more than half ofthese available on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisiteaniline biosynthetic activity for one or more genes in related ordistant species, including for example, homologues, orthologs, paralogsand nonorthologous gene displacements of known genes, and theinterchange of genetic alterations between organisms is routine and wellknown in the art. Accordingly, the metabolic alterations allowingbiosynthesis of aniline described herein with reference to a particularorganism such as E. coli can be readily applied to other microorganisms,including prokaryotic and eukaryotic organisms alike. Given theteachings and guidance provided herein, those skilled in the art willknow that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In some instances, such as when an alternative aniline biosyntheticpathway exists in an unrelated species, aniline biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesizeaniline.

Methods for constructing and testing the expression levels of anon-naturally occurring aniline-producing host can be performed, forexample, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production ofaniline can be introduced stably or transiently into a host cell usingtechniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to include one ormore aniline biosynthetic pathway encoding nucleic acids as exemplifiedherein operably linked to expression control sequences functional in thehost organism. Expression vectors applicable for use in the microbialhost organisms of the invention include, for example, plasmids, phagevectors, viral vectors, episomes and artificial chromosomes, includingvectors and selection sequences or markers operable for stableintegration into a host chromosome. Additionally, the expression vectorscan include one or more selectable marker genes and appropriateexpression control sequences. Selectable marker genes also can beincluded that, for example, provide resistance to antibiotics or toxins,complement auxotrophic deficiencies, or supply critical nutrients not inthe culture media. Expression control sequences can include constitutiveand inducible promoters, transcription enhancers, transcriptionterminators, and the like which are well known in the art. When two ormore exogenous encoding nucleic acids are to be co-expressed, bothnucleic acids can be inserted, for example, into a single expressionvector or in separate expression vectors. For single vector expression,the encoding nucleic acids can be operationally linked to one commonexpression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter.The transformation of exogenous nucleic acid sequences involved in ametabolic or synthetic pathway can be confirmed using methods well knownin the art. Such methods include, for example, nucleic acid analysissuch as Northern blots or polymerase chain reaction (PCR) amplificationof mRNA, or immunoblotting for expression of gene products, or othersuitable analytical methods to test the expression of an introducednucleic acid sequence or its corresponding gene product. It isunderstood by those skilled in the art that the exogenous nucleic acidis expressed in a sufficient amount to produce the desired product, andit is further understood that expression levels can be optimized toobtain sufficient expression using methods well known in the art and asdisclosed herein.

In some embodiments, the present invention provides a method forproducing aniline that includes culturing a non-naturally occurringmicrobial organism having an aniline pathway in which at least oneexogenous nucleic acid encoding an aniline pathway enzyme is expressedin a sufficient amount to produce aniline, under conditions and for asufficient period of time to produce aniline. The aniline pathwayincludes an aminodeoxychorismate synthase, an aminodeoxychorismatelyase, and a 4-aminobenzoate carboxylyase. In some embodiments, thepathway further includes a DAHP synthase. In some embodiments, thepathway further includes a 3-dehydroquinate synthase. A method forproducing aniline, includes culturing the non-naturally occurringmicrobial organism under conditions and for a sufficient period of timeto produce aniline. Moreover, the non-naturally occurring microbialorganism can be cultured in a substantially anaerobic culture medium.

Methods of the invention can include culturing a microbial organismhaving two exogenous nucleic acids each encoding an aniline pathwayenzyme. In some embodiments, the cultured microbial organism can includethree exogenous nucleic acids each encoding an aniline pathway enzyme.For example, the three exogenous nucleic acids can encode anaminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a4-aminobenzoate carboxylase. In some embodiments, the cultured microbialorganism can include four exogenous nucleic acids each encoding ananiline pathway enzyme. For example, the four exogenous nucleic acidscan encode a DAHP synthase, an aminodeoxychorismate synthase, anaminodeoxychorismate lyase, and a 4-aminobenzoate carboxylyase.

In still further embodiments, the cultured microbial organism caninclude five exogenous nucleic acids each encoding an aniline pathwayenzyme. For example, the five exogenous nucleic acids can encode a3-dehydroquinate synthase, a DAHP synthase, an aminodeoxychorismatesynthase, an aminodeoxychorismate lyase, and a 4-aminobenzoatecarboxylyase.

Any of the cultured organisms described above can have at least oneexogenous nucleic acid that is a heterologous nucleic acid.

In some embodiments, the present invention provides a method forproducing aniline, that includes culturing a non-naturally occurringmicrobial organism having an aniline pathway in which at least oneexogenous nucleic acid encoding an aniline pathway enzyme expressed in asufficient amount to produce aniline, under conditions and for asufficient period of time to produce aniline. In some embodiments, theaniline pathway includes an anthranilate synthase and an anthranilatedecarboxylase. In some embodiments, such an organism can further includea DAHP synthase. In some embodiments, such an organism can furtherinclude a 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, ashikimate dehydrogenase or a quinate/shikimate dehydrogenase, ashikimate kinase, a 3-phosphoshikimate-1-carboxyvinyltransferase, and achorismate synthase. In some embodiments, the cultured non-naturallyoccurring microbial organism is cultured in a substantially anaerobicculture medium.

In some embodiments, the above cultured microbial organism can includetwo exogenous nucleic acids each encoding an aniline pathway enzyme. Forexample, the two exogenous nucleic acids can encode an anthranilatesynthase and an anthranilate decarboxylase. In some embodiments, thecultured microbial organism can include three exogenous nucleic acidseach encoding an aniline pathway enzyme. For example, the threeexogenous nucleic acids encode a DAHP synthase, an anthranilate synthaseand an anthranilate decarboxylase. In still further embodiments, thecultured microbial organism can include four exogenous nucleic acidseach encoding an aniline pathway enzyme. For example, the four exogenousnucleic acids encode a 3-dehydroquinate synthase, a DAHP synthase, ananthranilate synthase and an anthranilate decarboxylase. Any of the atleast one exogenous nucleic acids can be provided as a heterologousnucleic acid.

Suitable purification and/or assays to test for the production ofaniline can be performed using well known methods. Suitable replicatessuch as triplicate cultures can be grown for each engineered strain tobe tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy) or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art(McCullough et al., J. Am. Chem. Soc. 79:628-630 (1957)).

Aniline can be separated from other components in the culture using avariety of methods well known in the art. Such separation methodsinclude, for example, extraction procedures as well as methods thatinclude continuous liquid-liquid extraction, pervaporation, membranefiltration, membrane separation, reverse osmosis, electrodialysis,distillation, crystallization, centrifugation, extractive filtration,ion exchange chromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the aniline producers can be cultured forthe biosynthetic production of aniline.

For the production of aniline, the recombinant strains are cultured in amedium with carbon source and other essential nutrients. It is highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic conditions can be applied byperforating the septum with a small hole for limited aeration. Exemplaryanaerobic conditions have been described previously and are well-knownin the art. Exemplary aerobic and anaerobic conditions are described,for example, in U.S. patent application Ser. No. 11/891,602, filed Aug.10, 2007. Fermentations can be performed in a batch, fed-batch orcontinuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch. Other sources of carbohydrates include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of aniline.

In addition to renewable feedstocks such as those exemplified above, theaniline microbial organisms of the invention also can be modified forgrowth on syngas as its source of carbon. In this specific embodiment,one or more proteins or enzymes are expressed in the aniline producingorganisms to provide a metabolic pathway for utilization of syngas orother gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Aceto genesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate an aniline pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the Wood-Ljungdahl enzymes or proteins absent in the hostorganism. Therefore, introduction of one or more encoding nucleic acidsinto the microbial organisms of the invention such that the modifiedorganism contains the complete Wood-Ljungdahl pathway will confer syngasutilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, aniline and any ofthe intermediate metabolites in the aniline pathway. All that isrequired is to engineer in one or more of the required enzyme or proteinactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of theaniline biosynthetic pathways. Accordingly, the invention provides anon-naturally occurring microbial organism that produces and/or secretesaniline when grown on a carbohydrate or other carbon source and producesand/or secretes any of the intermediate metabolites shown in the anilinepathway when grown on a carbohydrate or other carbon source. The anilineproducing microbial organisms of the invention can initiate synthesisfrom an intermediate, for example, chorismate, anthranilate,4-amino-4-deoxychorismate, or p-aminobenzoate.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding an anilinepathway enzyme or protein in sufficient amounts to produce aniline. Itis understood that the microbial organisms of the invention are culturedunder conditions sufficient to produce aniline. Following the teachingsand guidance provided herein, the non-naturally occurring microbialorganisms of the invention can achieve biosynthesis of aniline resultingin intracellular concentrations between about 0.1-200 mM or more.Generally, the intracellular concentration of aniline is between about3-150 mM, particularly between about 5-125 mM and more particularlybetween about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, ormore. Intracellular concentrations between and above each of theseexemplary ranges also can be achieved from the non-naturally occurringmicrobial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. publication2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicconditions, the aniline producers can synthesize aniline atintracellular concentrations of 5-10 mM or more as well as all otherconcentrations exemplified herein. It is understood that, even thoughthe above description refers to intracellular concentrations, anilineproducing microbial organisms can produce aniline intracellularly and/orsecrete the product into the culture medium.

In addition to the culturing and fermentation conditions disclosedherein, growth condition for achieving biosynthesis of aniline caninclude the addition of an osmoprotectant to the culturing conditions.In certain embodiments, the non-naturally occurring microbial organismsof the invention can be sustained, cultured or fermented as describedherein in the presence of an osmoprotectant. Briefly, an osmoprotectantrefers to a compound that acts as an osmolyte and helps a microbialorganism as described herein survive osmotic stress. Osmoprotectantsinclude, but are not limited to, betaines, amino acids, and the sugartrehalose. Non-limiting examples of such are glycine betaine, pralinebetaine, dimethylthetin, dimethylslfonioproprionate,3-dimethylsulfonio-2-methylproprionate, pipecolic acid,dimethylsulfonioacetate, choline, L-carnitine and ectoine. In oneaspect, the osmoprotectant is glycine betaine. It is understood to oneof ordinary skill in the art that the amount and type of osmoprotectantsuitable for protecting a microbial organism described herein fromosmotic stress will depend on the microbial organism used. The amount ofosmoprotectant in the culturing conditions can be, for example, no morethan about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM,no more than about 1.5 mM, no more than about 2.0 mM, no more than about2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no morethan about 7.0 mM, no more than about 10 mM, no more than about 50 mM,no more than about 100 mM or no more than about 500 mM.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of aniline includes anaerobic culture or fermentationconditions. In certain embodiments, the non-naturally occurringmicrobial organisms of the invention can be sustained, cultured orfermented under anaerobic or substantially anaerobic conditions.Briefly, anaerobic conditions refer to an environment devoid of oxygen.Substantially anaerobic conditions include, for example, a culture,batch fermentation or continuous fermentation such that the dissolvedoxygen concentration in the medium remains between 0 and 10% ofsaturation. Substantially anaerobic conditions also includes growing orresting cells in liquid medium or on solid agar inside a sealed chambermaintained with an atmosphere of less than 1% oxygen. The percent ofoxygen can be maintained by, for example, sparging the culture with anN₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of aniline. Exemplary growth proceduresinclude, for example, fed-batch fermentation and batch separation;fed-batch fermentation and continuous separation, or continuousfermentation and continuous separation. All of these processes are wellknown in the art. Fermentation procedures are particularly useful forthe biosynthetic production of commercial quantities of aniline.Generally, and as with non-continuous culture procedures, the continuousand/or near-continuous production of aniline will include culturing anon-naturally occurring aniline producing organism of the invention insufficient nutrients and medium to sustain and/or nearly sustain growthin an exponential phase. Continuous culture under such conditions caninclude, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include longer time periods of 1week, 2, 3, 4 or 5 or more weeks and up to several months.Alternatively, organisms of the invention can be cultured for hours, ifsuitable for a particular application. It is to be understood that thecontinuous and/or near-continuous culture conditions also can includeall time intervals in between these exemplary periods. It is furtherunderstood that the time of culturing the microbial organism of theinvention is for a sufficient period of time to produce a sufficientamount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of aniline can be utilized in, forexample, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

In addition to the above fermentation procedures using the anilineproducers of the invention for continuous production of substantialquantities of aniline, the aniline producers also can be, for example,simultaneously subjected to chemical synthesis procedures to convert theproduct to other compounds or the product can be separated from thefermentation culture and sequentially subjected to chemical conversionto convert the product to other compounds, if desired.

In some embodiments, methods for producing aniline include a step ofisolating aniline from the fermentation broth. This can be achieved bymeans of standard extraction, distillation, salt crystallizationtechniques, and combinations of these techniques and those describedabove. For a basic product such as aniline, a salt crystallization caninclude the formation of an acid salt of a Bronsted or Lewis acid.Exemplary acid salts include, without limitation, acetate, aspartate,benzoate, bicarbonate, carbonate, bisulfate, sulfate, chloride, bromide,benzene sulfonate, methyl sulfonate, phosphate, biphosphate, lactate,maleate, malate, malonate, fumarate, lactate, tartrate, borate,camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate,glucuronate, gluconate oxalate, palmitate, pamoate, saccharate,stearate, succinate, tartrate, tosylate and trifluoroacetate salts.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of aniline.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion or disruption strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene deletions or other functionalgene disruption, the growth selection pressures imposed on theengineered strains after long periods of time in a bioreactor lead toimprovements in performance as a result of the compulsory growth-coupledbiochemical production. Lastly, when gene deletions are constructedthere is a negligible possibility of the designed strains reverting totheir wild-type states because the genes selected by OptKnock are to becompletely removed from the genome. Therefore, this computationalmethodology can be used to either identify alternative pathways thatlead to biosynthesis of a desired product or used in connection with thenon-naturally occurring microbial organisms for further optimization ofbiosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat allow an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. publication2009/0047719, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

As disclosed herein, a nucleic acid encoding a desired activity of ananiline pathway can be introduced into a host organism. In some cases,it can be desirable to modify an activity of an aniline pathway enzymeor protein to increase production of aniline. For example, knownmutations that increase the activity of a protein or enzyme can beintroduced into an encoding nucleic acid molecule. Additionally,optimization methods can be applied to increase the activity of anenzyme or protein and/or decrease an inhibitory activity, for example,decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolutionis a powerful approach that involves the introduction of mutationstargeted to a specific gene in order to improve and/or alter theproperties of an enzyme. Improved and/or altered enzymes can beidentified through the development and implementation of sensitivehigh-throughput screening assays that allow the automated screening ofmany enzyme variants (for example, >10⁴). Iterative rounds ofmutagenesis and screening typically are performed to afford an enzymewith optimized properties. Computational algorithms that can help toidentify areas of the gene for mutagenesis also have been developed andcan significantly reduce the number of enzyme variants that need to begenerated and screened. Numerous directed evolution technologies havebeen developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical andbiotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press;Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al., ApplBiochem. Biotechnol 143:212-223 (2007)) to be effective at creatingdiverse variant libraries, and these methods have been successfullyapplied to the improvement of a wide range of properties across manyenzyme classes. Enzyme characteristics that have been improved and/oraltered by directed evolution technologies include, for example:selectivity/specificity, for conversion of non-natural substrates;temperature stability, for robust high temperature processing; pHstability, for bioprocessing under lower or higher pH conditions;substrate or product tolerance, so that high product titers can beachieved; binding (K_(m)), including broadening substrate binding toinclude non-natural substrates; inhibition (K_(i)), to remove inhibitionby products, substrates, or key intermediates; activity (kcat), toincreases enzymatic reaction rates to achieve desired flux; expressionlevels, to increase protein yields and overall pathway flux; oxygenstability, for operation of air sensitive enzymes under aerobicconditions; and anaerobic activity, for operation of an aerobic enzymein the absence of oxygen.

A number of exemplary methods have been developed for the mutagenesisand diversification of genes to target desired properties of specificenzymes. Such methods are well known to those skilled in the art. Any ofthese can be used to alter and/or optimize the activity of an anilinepathway enzyme or protein. Such methods include, but are not limited toEpPCR, which introduces random point mutations by reducing the fidelityof DNA polymerase in PCR reactions (Pritchard et al., J Theor. Biol.234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA),which is similar to epPCR except a whole circular plasmid is used as thetemplate and random 6-mers with exonuclease resistant thiophosphatelinkages on the last 2 nucleotides are used to amplify the plasmidfollowed by transformation into cells in which the plasmid isre-circularized at tandem repeats (Fujii et al., Nucleic Acids Res.32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006)); DNAor Family Shuffling, which typically involves digestion of two or morevariant genes with nucleases such as Dnase I or EndoV to generate a poolof random fragments that are reassembled by cycles of annealing andextension in the presence of DNA polymerase to create a library ofchimeric genes (Stemmer, Proc Natl Acad Sci USA 91:10747-10751 (1994);and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP),which entails template priming followed by repeated cycles of 2 step PCRwith denaturation and very short duration of annealing/extension (asshort as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998));Random Priming Recombination (RPR), in which random sequence primers areused to generate many short DNA fragments complementary to differentsegments of the template (Shao et al., Nucleic Acids Res 26:681-683(1998)).

Additional methods include Heteroduplex Recombination, in whichlinearized plasmid DNA is used to form heteroduplexes that are repairedby mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); andVolkov et al., Methods Enzymol. 328:456-463 (2000)); RandomChimeragenesis on Transient Templates (RACHITT), which employs Dnase Ifragmentation and size fractionation of single stranded DNA (ssDNA)(Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extensionon Truncated templates (RETT), which entails template switching ofunidirectionally growing strands from primers in the presence ofunidirectional ssDNA fragments used as a pool of templates (Lee et al.,J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide GeneShuffling (DOGS), in which degenerate primers are used to controlrecombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72 (2005); Gibbset al., Gene 271:13-20 (2001)); Incremental Truncation for the Creationof Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1base pair deletions of a gene or gene fragment of interest (Ostermeieret al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeieret al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-IncrementalTruncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which issimilar to ITCHY except that phosphothioate dNTPs are used to generatetruncations (Lutz et al., Nucleic Acids Res 29:E16 (2001)); SCRATCHY,which combines two methods for recombining genes, ITCHY and DNAshuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98:11248-11253(2001)); Random Drift Mutagenesis (RNDM), in which mutations made viaepPCR are followed by screening/selection for those retaining usableactivity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); SequenceSaturation Mutagenesis (SeSaM), a random mutagenesis method thatgenerates a pool of random length fragments using random incorporationof a phosphothioate nucleotide and cleavage, which is used as a templateto extend in the presence of “universal” bases such as inosine, andreplication of an inosine-containing complement gives random baseincorporation and, consequently, mutagenesis (Wong et al., Biotechnol.J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); andWong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling,which uses overlapping oligonucleotides designed to encode “all geneticdiversity in targets” and allows a very high diversity for the shuffledprogeny (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); NucleotideExchange and Excision Technology NexT, which exploits a combination ofdUTP incorporation followed by treatment with uracil DNA glycosylase andthen piperidine to perform endpoint DNA fragmentation (Muller et al.,Nucleic Acids Res. 33:e117 (2005)).

Further methods include Sequence Homology-Independent ProteinRecombination (SHIPREC), in which a linker is used to facilitate fusionbetween two distantly related or unrelated genes, and a range ofchimeras is generated between the two genes, resulting in libraries ofsingle-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460(2001)); Gene Site Saturation Mutagenesis™ (GSSM™), in which thestarting materials include a supercoiled double stranded DNA (dsDNA)plasmid containing an insert and two primers which are degenerate at thedesired site of mutations (Kretz et al., Methods Enzymol. 388:3-11(2004)); Combinatorial Cassette Mutagenesis (CCM), which involves theuse of short oligonucleotide cassettes to replace limited regions with alarge number of possible amino acid sequence alterations (Reidhaar-Olsonet al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al.Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis(CMCM), which is essentially similar to CCM and uses epPCR at highmutation rate to identify hot spots and hot regions and then extensionby CMCM to cover a defined region of protein sequence space (Reetz etal., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the MutatorStrains technique, in which conditional is mutator plasmids, utilizingthe mutD5 gene, which encodes a mutant subunit of DNA polymerase III, toallow increases of 20 to 4000-X in random and natural mutation frequencyduring selection and block accumulation of deleterious mutations whenselection is not required (Selifonova et al., Appl. Environ. Microbiol.67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)).

Additional exemplary methods include Look-Through Mutagenesis (LTM),which is a multidimensional mutagenesis method that assesses andoptimizes combinatorial mutations of selected amino acids (Rajpal etal., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly,which is a DNA shuffling method that can be applied to multiple genes atone time or to create a large library of chimeras (multiple mutations)of a single gene (Tunable GeneReassembly™ (TGR™) Technology supplied byVerenium Corporation), in Silico Protein Design Automation (PDA), whichis an optimization algorithm that anchors the structurally definedprotein backbone possessing a particular fold, and searches sequencespace for amino acid substitutions that can stabilize the fold andoverall protein energetics, and generally works most effectively onproteins with known three-dimensional structures (Hayes et al., Proc.Natl. Acad. Sci. USA 99:15926-15931 (2002)); and Iterative SaturationMutagenesis (ISM), which involves using knowledge of structure/functionto choose a likely site for enzyme improvement, performing saturationmutagenesis at chosen site using a mutagenesis method such as StratageneQuikChange (Stratagene; San Diego Calif.), screening/selecting fordesired properties, and, using improved clone(s), starting over atanother site and continue repeating until a desired activity is achieved(Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew.Chem. Int. Ed Engl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone orin any combination. Additionally, any one or combination of the directedevolution methods can be used in conjunction with adaptive evolutiontechniques, as described herein.

Example I Aniline Biosynthesis Using p-Aminobenzoate as the Precursor

This Example describes the generation of a microbial organism capable ofproducing aniline using chorismate as the precursor.

Escherichia coli is used as a target organism to engineer the pathwayusing the enzymes aminodeoxychorismate synthase, aminodeoxychorismatelyase, and 4-aminobenzoate carboxylyase as shown in FIG. 2. E. coliprovides a good host for generating a non-naturally occurringmicroorganism capable of producing aniline. E. coli is amenable togenetic manipulation and is known to be capable of producing variousproducts, like ethanol, acetic acid, formic acid, lactic acid, andsuccinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce aniline, nucleicacids encoding the enzymes utilized in the disclosed pathway, asdescribed previously, are expressed in E. coli to the desired extentusing well known molecular biology techniques (see, for example,Sambrook, supra, 2001; Ausubel supra, 1999; Roberts et al., supra,1989).

The native enzymes in E. coli can be modified or heterologous enzymescan be introduced to produce significant quantities of p-aminobenzoate.Further, 4-aminobenzoate carboxylyase activity can be incorporated intothe strain by introducing the appropriate genes, such as shdB, C and Dfrom Sedimentibacter hydroxybenzoicus. The genes are cloned into thepZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. The plasmid is transformed into the recombinant E. coli strainproducing p-aminobenzoate to express the proteins and enzymes requiredfor aniline synthesis from this metabolite.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of the pathwaygenes is corroborated using methods well known in the art fordetermining polypeptide expression or enzymatic activity, including, forexample, Northern blots, PCR amplification of mRNA, immunoblotting.Enzymatic activities of the expressed enzymes are confirmed using assaysspecific for the individually activities. The ability of the engineeredE. coli strain to produce aniline is confirmed using HPLC, gaschromatography-mass spectrometry (GCMS) or liquid chromatography-massspectrometry (LCMS).

Microbial strains engineered to have a functional aniline synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and in U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of aniline. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of aniline.Adaptive evolution also can be used to generate better producers of, forexample, the intermediate, chorismate or the product, aniline. Adaptiveevolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the aniline producer to further increaseproduction.

For large-scale production of aniline, the recombinant organism iscultured in a fermenter using a medium known in the art to supportgrowth of the organism under anaerobic conditions. Fermentations areperformed in either a batch, fed-batch or continuous manner. Anaerobicconditions are maintained by first sparging the medium with nitrogen andthen sealing culture vessel (e.g., flasks can be sealed with a septumand crimp-cap). Microaerobic conditions also can be utilized byproviding a small hole for limited aeration. The pH of the medium ismaintained at a pH of 7 by addition of an acid, such as H₂SO₄. Thegrowth rate is determined by measuring optical density using aspectrophotometer (600 nm), and the glucose uptake rate by monitoringcarbon source depletion over time. Byproducts such as undesirablealcohols, organic acids, and residual glucose can be quantified by HPLC(Shimadzu) with an HPX-087 column (BioRad), using a refractive indexdetector for glucose and alcohols, and a UV detector for organic acids(Lin et al., Biotechnol Bioeng. 90:775-779 (2005)).

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties, includingGenBank and GI number publications, are hereby incorporated by referencein this application in order to more fully describe the state of the artto which this invention pertains. Although the invention has beendescribed with reference to the examples provided above, it should beunderstood that various modifications can be made without departing fromthe spirit of the invention.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

What is claimed is:
 1. A method for producing aniline comprising: (a)culturing a non-naturally occurring Escherichia coli under conditionsand for a sufficient period of time to produce aniline, wherein thenon-naturally occurring Escherichia coli comprises an aniline pathway,the aniline pathway comprising an anthranilate synthase, a3-dehydroquinate synthase, a 3-dehydroquinate dehydratase, a shikimatedehydrogenase or a quinate/shikimate dehydrogenase, a shikimate kinase,a 3-phosphoshikimate-1-carboxyvinyltransferase, a chorismate synthase,and an anthranilate decarboxylase, wherein the non-naturally occurringEscherichia coli comprises at least two exogenous nucleic acids encodingat least two enzymes of the aniline pathway selected from the groupconsisting of the 3-dehydroquinate synthase, the anthranilate synthase,the 3-dehydroquinate dehydratase, the shikimate dehydrogenase or thequinate/shikimate dehydrogenase, the shikimate kinase, the3-phosphoshikimate-1-carboxyvinyltransferase, and the chorismatesynthase, wherein the anthranilate decarboxylase in the non-naturallyoccurring Escherichia coli is endogenous, and wherein the anilinepathway enzymes are expressed in a sufficient amount to produce aniline;and (b) isolating aniline.
 2. The method of claim 1, wherein the anilinepathway further comprises a 3-deoxy-D-arabino-heptulosonicacid-7-phosphate (DAHP) synthase.
 3. The method of claim 1, wherein saidnon-naturally occurring microbial organism Escherichia coli is in asubstantially anaerobic culture medium.
 4. The method of claim 2,wherein said non-naturally occurring Escherichia coli comprises threeexogenous nucleic acids encoding three enzymes of the aniline pathwayselected from the group consisting of the 3-dehydroquinate synthase, theanthranilate synthase, the 3-dehydroquinate dehydratase, the shikimatedehydrogenase or the quinate/shikimate dehydrogenase, the shikimatekinase, the 3-phosphoshikimate-1-carboxyvinyltransferase, the chorismatesynthase, and the DAHP synthase.
 5. The method of claim 4, wherein saidthree exogenous nucleic acids encode the DAHP synthase, the anthranilatesynthase and the 3-dehydroquinate synthase.
 6. The method of claim 2,wherein said non-naturally occurring Escherichia coli comprises fourexogenous nucleic acids encoding four enzymes of the aniline pathwayselected from the group consisting of the 3-dehydroquinate synthase, theanthranilate synthase, the 3-dehydroquinate dehydratase, the shikimatedehydrogenase or the quinate/shikimate dehydrogenase, the shikimatekinase, the 3-phosphoshikimate-1-carboxyvinyltransferase, the chorismatesynthase, and the DAHP synthase.
 7. The method of claim 6, wherein saidfour exogenous nucleic acids encode the DHAP synthase, the anthranilatesynthase, and two enzymes of the aniline pathway selected from the groupconsisting of the 3-dehydroquinate synthase, the 3-dehydroquinatedehydratase, the shikimate dehydrogenase or the quinate/shikimatedehydrogenase, the shikimate kinase, the3-phosphoshikimate-1-carboxyvinyltransferase, and the chorismatesynthase.
 8. The method of claim 1, wherein said at least two exogenousnucleic acids are heterologous nucleic acids.